The ability of Histoplasma capsulatum (Hc), to persist within mammalian tissue after initial infection is fundamental to the pathogenesis of disease. Most individuals recover from primary infection, however, with impairment of cellular immunity, may develop progressive disease by reactivation of dormant foci. Persistence within the host requires evasion of innate and adaptive immune mechanisms. The long term goal of the research is to better understand the biology of latency and reactivation allowing the development of therapeutic interventions to enhance organism eradication. The underlying issue to be addressed in this proposal is the elucidation of the mechanisms that allow organism survival within infected tissues. Initial adaptation of the pathogen is important in establishing infection in the face of the innate immune onslaught. The host response controls but does not eradicate Hc. A second mechanism of defense used by the host is to wall off invading organisms and limit the availability of important nutrients. The central hypothesis is that adaptations to nutritional stress are critical for Hc's ability to persist within infected tissues. The hypothesis is formulated on the basis of preliminary data where the Hc transcriptome was analyzed and changes associated with in vivo infection identified. Prominent among the changes were upregulation of genes involved in lipid metabolism. Silencing one of these genes, encoding a putative acyl-CoA synthetase (ACS), resulted in a strain which, while able to establish infection in a similar manner to controls, was more rapidly cleared from infected animals and was unable to reactivate. The mechanism by which this strain is impaired in its ability to persist will be tested by pursuing 2 specific aims: 1) Determine the importance of specific ACS activities on growth and latency; and 2) Determine the mechanisms by which genes of the fatty acid degradation pathway impact the ability to persist and reactivate. Under the first aim, studies will confirm the designation of the gene encoding Orf 12k15 as an ACS by determining the activity of Orf 12k15 protein biochemically in Saccharomyces cerevisiae (Sc) and Hc and by functional complementation in Sc. Using RNA interference (RNAi) and microarray analysis, it will be determined if impaired persistence results from an inability to utilize fatty acids as an energy source, altered gene regulation or impaired protein acylation. Aim 2 will elicit the mechanisms impairing the ability of the suppressed strain to persist. In vitro studies will examine the ability of silenced strains to grow under conditions of nutrient deprivation and hypoxia, to tolerate noxious stimuli such as nitric oxide, and to adapt to stationary phase growth. Studies using animal models with compromised adaptive responses will dissect the role of impaired ability to establish infection compared to the ability to persist to latency. Other in vivo studies will determine if the failure to establish latency is due to altered trafficking within populations of phagocytes, due to an inability to disseminate, or due to failure to modulate the immune response. The proposed research will be significant in enhancing an understanding of the biology of latency of the organism. PUBLIC HEALTH RELEVANCE: POTENTIAL IMPACT ON VETERANS HEALTH CARE. Reactivation of dormant infection is the most common mechanism of clinically evident histoplasmosis and often arises in the context of a weakened immune system. Reactivation histoplasmosis causes significant morbidity among the veterans population. Better understanding of the molecular determinants of reactivation may allow alternative approaches to prevent clinical disease.